This invention relates generally to peptides having unnatural amino acids and to the preparation of new unnatural amino acids, which may be derived from diamino acids, such as Lys, Orn, Dpr and Dbu. More particularly, it relates to GnRH analogs having such unnatural amino acids which can be prepared either in such fully assembled peptides or for incorporation into such peptides as a part of the usual chain elongation synthesis process.
In one more particular aspect, the present invention relates to peptides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone and also to peptides which promote the release of such steroids, as well as to methods of promoting or preventing ovulation.
The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. In particular, follicle stimulating hormone (FSH) and luteinizing hormone (LH), sometimes referred to as gonadotropins or gonadotropic hormones, are released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to produce testosterone in the testes and progesterone and estrogen in the ovaries, and they also regulate the production and maturation of gametes.
The release of a hormone by the anterior lobe of the pituitary gland usually requires a prior release of another class of hormones produced by the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH, and this hormone is referred to herein as GnRH although it has also been referred to as LH-RH and as LRF. GnRH was isolated and characterized as a decapeptide some 20 years ago, and it was found that analogs of GnRH having a D-isomer instead of Gly in the 6-position, such as [D-Ala6]-GnRH (U.S. Pat. No. 4,072,668) having the following formula:
pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2,
have greater binding strength to the receptor and greater biological potency than the native hormone.
Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for GnRH, as represented above, is in accordance with conventional representation of peptides where the amino terminus appears to the left and the carboxyl terminus to the right. The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine at the C-terminus has been replaced with an amino group(NH2) i.e. the C-terminus is amidated. The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid, e.g. pGlu is pyroglutamic acid, Glu is glutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Nle is norleucine, Orn is ornithine, Arg is arginine, Har is homoarginine, Pro is proline, Sar is sarcosine, Phe is phenylalanine, Ala is alanine, Val is valine, Nva is norvaline, Ile is isoleucine, Thr is threonine, Lys is lysine, Asp is aspartic acid, Asn is asparagine, Gln is glutamine, and Met is methionine. Except for glycine, amino acids of the peptides of the invention are of the L-configuration unless noted otherwise.
There are reasons for desiring to prevent ovulation in female mammalians, and the administration of GnRH analogs that are antagonistic to the normal function of GnRH have been used to suppress or delay ovulation. For this reason, analogs of GnRH which are antagonistic to GnRH are being investigated for their potential use as a contraceptive or for regulating conception periods. GnRH antagonists may also be used for the treatment of precocious puberty and endometriosis. The GnRH superagonists can also be used for these purposes. Such antagonists have also been found useful to regulate the secretion of gonadotropins in male mammals and can be employed to arrest spermatogenesis, e.g. as male contraceptives for treatment of male sex offenders, and for treatment of prostatic hypertrophy. More specifically, GnRH antagonists can be used to treat steroid-dependent tumors, such as prostatic and mammary tumors, and for the control of the timing of ovulation for in vitro fertilization. In the female, they can also be used to treat hirsutism.
On the other hand, GnRH agonists function in the same manner as GnRH in promoting the release of LH and FSH, and agonists which exhibit greater biopotency and/or longer duration of action are considered valuable.
In one aspect, it is desired to provide improved peptides which either are strongly antagonistic to endogenous GnRH and prevent secretion of LH and FSH and the release of steroids by the gonads of mammals or are strong agonists of GnRH. Of particular interest are compounds which are more effective in vivo when administered orally.
The present invention provides unnatural amino acids that can be prepared de novo or by modifying a previously prepared peptide, but which are preferably prepared as a part of a protected peptide-resin, containing a desired overall sequence which includes one or more amino acid residues having a side chain amino group which is to be modified. The resultant preferred amino acids have a side chain which contains either a modified guanidino group or a guanidino equivalent or a derivative that is obtained by further elucidation of a modified guanidino group, as set forth hereinafter, and most preferably it contains a triazole moiety.
In another particular aspect, the invention provides peptides which inhibit the release of gonadotropins in mammalians, including humans, and it also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians. The invention also provides improved GnRH analogs which are strong agonists of GnRH and can be used to promote the reproduction processes of mammalians. As mentioned above, these GnRH antagonists may be used to inhibit the production of gonadotropins and sex hormones under various circumstances, including precocious puberty, hormone dependent neoplasia, dysmenorrhea, endometriosis and steroid-dependent tumors.
The invention provides unnatural amino acids having the following formula U*: 
where W is (CH2)n or 
n is an integer from 1 to 6 and is preferably 1,2,3 or 4; j is 1,2 or 3; Y=Nxe2x80x94CN, Nxe2x80x94CONHR9, O, S or CHxe2x80x94NO2, where R9 is H, Ac, alkyl (preferably C1 to C4), naphthyl, pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl or imidazolyl, which alkyl and cyclic groups are unsubstituted or substituted (preferably by chloro, fluoro, bromo, amino, nitro, alkyl (C1 to C4) and alkoxy (C1 to C4)); Xxe2x95x90NH, O, N3, M1xe2x80x94(CHq)pxe2x80x94M2 or M1xe2x80x94(CH2)pxe2x80x2xe2x80x94M2(CH2)pxe2x80x3xe2x80x94M3, where M1 is NR10, N, O or CHR3 wherein R3 is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl; q=1 or 2; p, pxe2x80x2 and pxe2x80x3 are integers between 0 and 6; R10 is H, methyl, ethyl, propyl, phenyl or substituted phenyl (preferably by Cl, F, NO2 or NH2); and M2 and M3xe2x95x90M1, COOH, CONH2, COOR3 or CN (preferably X is NH or O); R1xe2x95x90H, alkyl (preferably C1 to C6 and most preferably C1 to C4), modified alkyl (preferably C1 to C5, the terminal carbon of which is either substituted with NH2, OH, Cl, Br or F or is replaced with CF3 or CF2CF3), alkenyl (preferably C2 to C4), such as CH2CHxe2x95x90CHR3, alkynyl (preferably C2 to C4), such as CH2Cxe2x89xa1CR3, aryl such as benzyl, tolyl, p-amino-benzyl (anilinyl) and pCl-benzyl or a direct bond to X; R2xe2x95x90R1, OH, NH2, NHR1, heterocycle (preferably as illustrated hereinafter) or desR2, with R2 being desR2 when Xxe2x95x90N3. Optionally R2 and X can be interconnected, or R1 and R2 can be connected to each other via a branched or unbranched methylene bridge of type xe2x80x94(CH2)mxe2x80x94 or xe2x80x94(CH2)mxe2x80x94Mxe2x80x94(CH2)mxe2x80x2xe2x80x94. In such an R1xe2x80x94R2 moiety, m and mxe2x80x2 are integers from 1 to 6 and preferably from 1 to 3; and Mxe2x95x90NH, O, S or CHR4, wherein R4 is lower alkyl or aryl and is preferably methyl, ethyl, propyl, phenyl or pcl-phenyl, with M preferably being O or S. Most preferably, when R1 and R2 are interconnected, they form a 5, 6, or 7-member heterocyclic ring with the xe2x80x9cNxe2x80x94Cxe2x80x94Xxe2x80x9d portion of the formula U*. If desired to form a cyclic peptide, XR2 can contain a part of another diamino acid within the same peptide, e.g., the omega amino group of the 5-position residue can be so linked to such an unnatural amino acid residue in the 8-position.
Modification of the specified primary amino function of a given amino acid or peptide is carried out by treatment of either the appropriately protected peptide or the amino acid with an appropriate reagent(s). Peptides or amino acids where Y is Nxe2x80x94CN (herein referred to as cyanoguanidines) are prepared by reaction of an amino group with diphenyl cyanocarbonimidate (I): 
wherein xe2x80x9cQxe2x80x9d is used to broadly represent either the major portion of a peptide or an amino acid having a primary amino group (such as the amino acid which is depicted above) as a part of formula U*.
The peptide or amino acid having the N-substituted-Nxe2x80x2-cyano-O-phenylisourea moiety (II) can then be either isolated or further functionalized by reaction with a second nucleophile HXR2 to produce cyanoguanidine-containing peptides or amino acids having the formula (III): 
For example, where HXR2xe2x95x90H2Nxe2x80x94CH2-pyridine, the result is: 
This group may also be referred to (IUPAC nomenclature) as N-g-cyano-N-gxe2x80x2-3-methylpyridylguanidino.
Such compounds can be hydrolyzed under acidic conditions to produce compounds which are also biopotentxe2x80x94for example: 
The hydrolyzed versions, referred to herein as including the N-gxe2x80x2-amido group, can also be synthesized directly by reacting phosgene derivates with moieties having a guanidino function.
If HXR2 is an amino group of another peptide or protein, one will obtain a peptide-peptide dimer or peptide-protein dimer conjugated via the cyanoguanidine moiety. If HXR2 is the N-terminal primary amino group or the side chain amino group of another amino acid in the same peptide, one will obtain a cyclic peptide (IV) linked via the cyanoguanidine moiety: 
wherein Q1 and Q2 represent the remainders of two amino acid residues in the same peptide. Cyclization via the cyanoguanidine derivative is preferably effected while a part of the peptidoresin, as opposed to subsequently cyclizing the linear peptide.
A special case arises when xe2x80x94XR2 contains a second nucleophilic site and X has the general form: M1xe2x80x94(CHq)pxe2x80x94M2 or M1xe2x80x94(CH2)pxe2x80x2xe2x80x94M2xe2x80x94(CH2)pxe2x80x3xe2x80x94M3, where M1, M2 and M3 are individually NH, N, O or CHR3, with p, pxe2x80x2, pxe2x80x3 being 0,1,2 or 3 and q being 1 or 2. Examples of such nucleophiles include H2NNH2, CH3HNNH2, CH3HNNHCH3, H2NOH, and H2Nxe2x80x94CH2xe2x80x94CH2OH. In this case, the cyanoguanidine moiety that is formed can be converted into the corresponding heterocycle (V) which forms from the initial intermediate by reaction of the omega amino group with the cyano group such as: 
For example, where xe2x80x94XR2xe2x95x90xe2x80x94HNNH2, 
Furthermore, where xe2x80x94XR2xe2x95x90xe2x80x94CH3NNHCH3
which may then undergo hydrolysis, as indicated above.
Where XR2 contains a carboxylic acid group or the equivalent, particularly a carboxylic ester or carboxylic amide, a heterocyclic moiety, such as a saturated pyrimidine-like moiety (VI), is formed, by reaction of the carboxylic group with the secondary amino group (R1), when M1 is N, and similar 6-membered heterocyclic moieties are formed when M1 is O or S. For example, R2 may be M1xe2x80x94(CH2)pxe2x80x94M2 with M2xe2x95x90COOH, COOCH3 or CONH2 and p being an integer between 1 and 4. For instance in such a case where an aliphatic carboxylic acid group is present and p=2: 
If R2 includes an ortho-substituted aromatic carboxylic acid, e.g. benzoic acid (q=1 and p=6), the corresponding quinazoline-like species (VII) is formed: 
Such benzoic acid may be further substituted, and such substitutions may in any of the other 4 ring positions, as shown, creating the corresponding substituted quinazoline-like moiety which is considered to be equivalent to the unsubstituted. Xxe2x80x2 may be H, Cl, Br, F, NHCH3 or SCH3, and R7 and R8 may be H, CH3 or CH2CH3.
The molecules wherein X=N3 and R2 is desR2 (i.e. deleted) are useful for photolabeling because of the activity of the xe2x80x94N3 group and are formed by reacting the moiety (II) with sodium azide (NaN3).
Peptides wherein Y is O (herein referred to as ureas) or S (referred to as thioureas) are prepared by the well known procedure in which the desired side chain amino group is treated with an appropriate isocyanate or thioisocyanate to obtain such ureas or thioureas. 
Peptides or amino acids wherein Y is CHxe2x80x94NO2 (herein referred to as diaminonitroethylenes) are prepared by conversion of the corresponding urea to a carbodiimide: 
followed by treatment with nitromethane anion (prepared by the action of sodium hydride on nitromethane in dry DMF) as disclosed generally in F. Meimas, et al., Synthesis, 509-510 (1985): 
An alternative synthesis that may be used is as follows: 
Generally, in accordance with the present invention, peptides are synthesized which are antagonists or agonists of GnRH. Antagonists strongly inhibit the secretion of gonadotropins by the pituitary gland of mammals, including humans, and/or inhibit the release of steroids by the gonads. Agonists strongly promote such secretion or release. These peptides are analogs of GnRH containing one or more unnatural amino acids of the formula U* in the 3-position, the 5-position, the 6-position and/or the 8-position. When U* is in the 3- and/or 6-position, it is always in the form of a D-isomer; whereas when U* is in the 5- and/or 8-position, it is always in the form of an L-isomer. An antagonist should have a 1-position substitution, such as dehydropro or xcex2-(1- or 2-naphthyl)-D-alanine (hereinafter xcex2-D-1NAL or xcex2-D-2NAL), a 2-position substitution in the form of a modified D-Phe and a 3-position substitution, preferably in the form of substituted or unsubstituted D-Trp, D-3PAL, xcex2-D-NAL or the residue of a D-isomer amino acid U*. The 5-position may be occupied by (a) Tyr, (b) a halogenated or methylated Phe or Tyr, (c) Arg, (d) Lys in which the side chain amino group is acylated by 3-carboxypyridine (nicotinic acid) or by 2 or 4-carboxypyridine, i.e. Lys(cpd), preferably Lys(3cpd) which is also referred to as Lys(Nic), (e) His or (f) the residue of an L-isomer amino acid U*. Agonists have a 6-position substitution which is the residue of the D-isomer U*, and the antagonists may have a D-isomer U* or such a substituted or acylated D-Lys in the 6-position. Instead of Leu in the 7-position, both may have Nle, NML, Phe, Nva, Met, Tyr, Trp or PAL, of which the Phe or Trp may be substituted. The antagonists may also have an optional substitution in the 8-position, which preferably may be the L-isomer U* or isopropyl Lys, i.e., ILys or Lys(Ipr) wherein the side chain amino group is substituted by isopropyl, and a substitution in the 10-position such as D-Ala. At least one residue of a D-isomer amino acid of the formula U* is most preferably present in each peptide of the invention.
Modified D-Phe in the 2-position provides increased antagonistic activity as a result of the specific modifications present in the benzene ring. Single substitutions for hydrogen in the ring are preferably made in the para- or 4-position, but might be in either the 2- or 3-position also; the substitutions are selected from chloro, fluoro, bromo, methyl, methoxy and nitro, with chloro, fluoro and nitro being preferred. Dichloro substitutions are in the 2,4 or 3,4 positions in the ring. The xcex1-carbon atom may also be methylated, e.g. (Cxcex1Me/4Cl)Phe. The 1-position substituent is preferably modified so that its xcex1-amino group contains an acyl group, such as formyl(For), acetyl(Ac), acrylyl(Acr), vinylacetyl(Vac) or benzoyl(Bz), with acetyl and acrylyl being preferred and with acetyl being most preferred. PAL and D-PAL represent the L- and D-isomers of pyridylalanine where the xcex2-carbon of Ala is linked to the 2-, 3- or 4-position, preferably to the 3-position, on the pyridine ring. When xcex2-D-NAL is present in the 1-position and R5 is not Arg, a hydrophilic D-amino acid residue, such as 4NH2-D-Phe, 4-guanidino-D-Phe, D-His, D-Lys, D-Orn, D-Arg, D-Har(Homoarginine) or D-PAL is preferably present in the 6-position if U* is not present. When dehydropro is present in the 1-position, D-PAL or a D-isomer of a lipophilic amino acid, such as D-Trp, D-Phe, For-D-Trp, NO2-D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val, D-Ala, dialkyl Arg, dialkyl Har, D-Ser(OtBu), xcex2-D-NAL or (imBzl)D-His is preferably in the 6-position, if U* is not present.
These GnRH analogs are very soluble at a pH just below physiological pH, i.e. about 4.5 to about 6, and thus can be formulated and administered in concentrated form, greatly facilitating administration at a pH of about 5 to 7.4 which is presently preferred. The agonists are substantially more potent than native GnRH in effecting release of LH and FSH. The antagonists inhibit ovulation of female mammals when administered at low levels at proestrus and are also effective to cause resorption of fertilized eggs if administered shortly after conception. The antagonists are also effective for the contraceptive treatment of male mammals and the treatment of steroid-dependent tumors. Certain of the antagonists are surprisingly long-acting in their suppression of LH levels following administration, and certain have a particularly low side effect in respect of histamine release.
As previously mentioned, the unnatural amino acids (which can be L- or D-isomers) are represented by the formula U*: 
wherein W, X, Y, R1 and R2 are as defined previously, and there is at least one such residue (preferably a D-isomer) in each peptide of the invention.
More specifically, the GnRH antagonists of the present invention are represented by the following Formula (F1):
G-AA1-(A)D-Phe-AA3-Ser-AA5-AA6-AA7-AA8-Pro-AA10 
wherein G is hydrogen or an acyl group having 7 or less carbon atoms; AA1 is dehydropro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or P-D-NAL; A is H, Cl, F, NO2, CH3, OCH3, Cxcex1Me/4Cl, Cl2 or Br; B is H, NO2, NH2, OCH3, F, Cl, Br, CH3, NinFor or NinAc; AA3 is U*, D-PAL, 6-D-NAL or (B)D-Trp; AA5 is U*, Tyr, (C)Arg, Lys(cpd), Orn(cpd), Dbu(cpd), Dpr(cpd), (A)Phe, (3I)Tyr or His; AA6 is U*, xcex2-D-NAL, (B)D-Trp, (Axe2x80x2)D-Phe, (D)D-Orn, (D)D-Lys, (D)D-Dbu, (D)D-Dpr, D-Har, D-Tyr, (E)D-His, D-PAL, (C)D-Arg or a suitable lipophilic D-isomer; Axe2x80x2 is A, NH2, NHCH3 or gua; C is H or lower alkyl; D is G, cpd or an aryl group; E is H, imBzl or dinitrophenol; AA7 is Nle, Leu, NML, (A)Phe, Met, Nva, Tyr, (B)Trp or PAL; AA8 is U*, (Cxe2x80x2)Arg, (Cxe2x80x2)Har or ILys; Cxe2x80x2 is H or di-lower alkyl; AA10 is D-Ala-NH2, Gly-NH2, AzaGly-NH2 or NH(R); R is lower alkyl, preferably CH2CH3; and U* is as defined above. When AA1 is xcex2-D-NAL and AA5 is not Arg, then AA6 is preferably U*, 4-NH2-D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D-Arg.
By dehydropro is meant 3,4 dehydroproline, C5H7O2N. By xcex2-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the xcex2-carbon atom, i.e., also 3-D-NAL. Preferably xcex2-D-2NAL is employed wherein the attachment to naphthalene is at the 2-position on the ring structure; however, xcex2-D-1NAL may also be used. The preferred 1-position residues are xcex2-D-NAL, substituted D-Phe and optionally substituted D-Trp. PAL represents alanine which is substituted by pyridyl on the xcex2-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When substituted D-Trp is employed, single substitutions for hydrogen are preferably made in either the 5- or 6-position, which are selected from chloro, fluoro, bromo, methyl, amino, methoxy and nitro, with chloro, fluoro and nitro being preferred. Alternatively, the indole nitrogen may be acylated, e.g. with formyl (NinFor- or 1For-) or with acetyl. D-3PAL, NinFor-D-Trp and 6NO2-D-Trp are the preferred residues for the 3-position although D-Trp is also often used. When U* is not in the 5-position, Tyr, Arg or Lys(cpd) preferably is present. By NML is meant Nxcex1CH3-L-Leu. By Dbu is meant alpha, gamma diamino butyric acid, and by Dpr is meant xcex1,xcex2 diamino propionic acid. By Aph is meant 4NH2Phe; aminohomophenylalanine and aminohomohomophenylalanine are considered to be equivalents thereof. Instead of being in the para- or 4-position, the amino substitution may also be in the meta or ortho position. When dehydropro is present in the 1-position, Tyr or U* is preferably present in the 5-position and a lipophilic residue is in the 6-position. By 4-gua-D-Phe is meant a residue of D-Phe having guanidine substituted in the para-position. By AzaGly-NH2 is meant NHNHCONH2. The guanidino group of an Arg residue in the 5- or 6-position may be substituted by lower alkyl, i.e. 1 to 4 carbon atoms, e.g., propyl(Pr). When D-Lys, D-Dbu, D-Dpr or D-Orn is present in the 6-position and it is not a part of an unusual amino acid U*, its side-chain-amino group may be acylated by an acyl group which may be aliphatic, heterocyclic or aromatic, e.g. nicotinic acid, or may be substituted by an aryl group having not more than 1 phenyl ring. When U* is not present in the 6-position, it is preferably D-PAL or D-Lys(cpd). The 7-position residue is preferably Leu, NML, Nle or Phe. If the 8-position residue is not U*, it is preferably ILys.
More specifically the GnRH agonists of the invention are represented by the following Formula (F2): pGlu-His-Trp-Ser-Tyr-U*-AA7-Arg-Pro-AA10, wherein U*, AA7 and AA10 are as defined hereinbefore. Preferably AA7 is Leu or NML, AA10 is NHCH2CH3 and U* includes a residue of amino-substituted Phe (or its equivalent, e.g homophenylalanine).
Overall, the invention thus provides GnRH analogs having the Formula (F3):
G-AA-AA2-AAxe2x80x2-Ser-AA5-AA6-AA7-AA8-Pro-AA10 wherein AA is pGlu or AA1; AA2 is His or (A)D-Phe; AAxe2x80x2 is Trp or AA3;
and all others are as defined hereinbefore.
One preferred subgenus of GnRH antagonists has the following formula:
Ac-AA1-(A)D-Phe-AA3-Ser-AA5-AA6-AA7-AA8-Pro-AA10 wherein AA1 is (A)D-Phe, (B)D-Trp or xcex2-D-NAL; A is H, 4Cl,4F, 4NO2, 4CH3, 4OCH3, Cxcex1Me/4Cl, 2,4Cl2 or 4Br; B is H, 6NO2, 6NH2, 6OCH3, 6F, 6Cl, 6Br, 6CH3, NinFor or NinAc; AA3 is U*, D-PAL, xcex2-D-NAL or (B)D-Trp; AA5 is U*, Lys(cpd) or Tyr; AA6 is U*, xcex2-D-NAL, 4NH2D-Phe, (B)D-Trp, D-Lys(cpd), D-PAL or D-Arg; AA7 is Nle, Leu, NML or Phe; AA8 is U*, ILys, or Arg; AA10 is D-Ala-NH2, Gly-NH2, NHNHCONH2 or NH(R); R is lower alkyl; and U* is either 
where W is (CH2)n or 
xe2x80x83n is an integer from 1 to 4; j is 1,2 or 3; X is NH or O; Y is Nxe2x80x94CN or Nxe2x80x94CONHR9 where R9 is H or lower alkyl; R2 is lower alkyl, cyclohexyl, phenyl, pyridyl, methyl pyridyl or histaminyl; or 
where W is defined as above and R11 is H or an acyl radical having 1 to 6 carbon atoms; provided, however, that at least one of AA3, AA5, AA6 and AA8 is U*.
Another preferred subgenus of GnRH antagonists has the following formula:
Ac-AA1-(A)D-Phe-U*-Ser-AA5-AA6-AA7-AA8-Pro-AA10 wherein AA1 is (A)D-Phe, (B)D-Trp or xcex2-D-NAL; A is H, 4Cl,4F, 4NO2, 4CH3, 4OCH3, Cxcex1Me/4Cl, 2,4Cl2 or 4Br; B is H, 6NO2, 6NH2, 6OCH3, 6F, 6Cl, 6Br, 6CH3, NinFor or NinAc; AA5 is Lys(cpd) or Tyr; AA6 is xcex2-D-NAL, 4NH2D-Phe, (B)D-Trp, D-Lys(cpd), D-PAL or D-Arg; AA7 is Nle, Leu, NML or Phe; AA8 is ILys or Arg; AA10 is D-Ala-NH2, Gly-NH2, NHNHCONH2 or NH(R); R is lower alkyl; and U* is either 
where W is (CH2)n or 
xe2x80x83n is an integer from to 4; j is 1,2 or 3 (preferably 1); X is NH or O; Y is Nxe2x80x94CN or Nxe2x80x94CONHR where R9 is H or lower alkyl (C1-C3), preferably H; R2 is lower alkyl (C1-C6), cyclohexyl, phenyl, pyridyl, methyl pyridyl or histaminyl; or 
where W is defined as above and R11 is H or an acyl radical having 1 to 3 carbon atoms, preferably acetyl.
Still another preferred subgenus of GnRH antagonists has the formula:
Ac-xcex2-D-2NAL-(4Cl)D-Phe-U*-Ser-Tyr-D-3PAL-Leu-Lys(isopropyl)-Pro-D-Ala-NH2, wherein U* is a D-isomer having the formula either 
where R2 is lower alkyl.
Yet another preferred subgenus of GnRH antagonists has the formula:
Ac-xcex2-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-U*-U*-AA7-Lys (isopropyl)-Pro-AA10 wherein AA7 is Leu or NaCH3Leu; AA10 is D-Ala-NH2, Gly-NH2, or NHCH2CH3; and U* is either 
where R2 is lower alkyl, pyridyl or methyl, pyridyl;
provided, however, that U* in the 6-position is always a D-isomer.
An additional preferred subgenus of GnRH antagonists has the formula:
Ac-xcex2-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-U*-U*-AA7-Lys (isopropyl)-Pro-AA10 wherein AA7 is Leu or NaCH3-Leu; AA10 is D-Ala-NH2, Gly-NH2 or NHCH2CH3; and U* is 
where W is (CH2)n or 
xe2x80x83n is an integer from 1 to 6; and R11 is H or an acyl radical having 1 to 6 carbon atoms; provided, however, U* in the 6-position is a D-isomer.
One more preferred subgenus of GnRH antagonists has the formula:
Ac-xcex2-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA5-AA6-Leu-Lys (isopropyl)-Pro-D-Ala-NH2 wherein AA5 is U* or Lys(carboxypyridine); AA6 is U* or D-Lys(carboxypyridine); and U* is 
where W is (CH2)n and n is 4; provided, however, that either AA5 is U* or AA6 is U*, with AA6 always being a D-isomer.
The peptides of the present invention can be synthesized by classical solution synthesis, but are preferably synthesized by a solid phase technique. A chloromethylated resin or a hydroxymethylated resin may be used; however, a methylbenzhydrylamine(MBHA) resin, a benzhydrylamine (BHA) resin or some other suitable resin known in the art which directly provides a C-terminal amide or substituted amide upon cleavage is preferably employed when such a C-terminus is desired. For example, peptides having a substituted amide at the C-terminus are preferably synthesized using an N-alkylamino methyl-resin as taught in U.S. Pat. No. 4,569,967, issued Feb. 11, 1986. Solid phase synthesis is conducted in a manner to stepwise add amino acids in the chain in the manner set forth in detail in the U.S. Pat. No. 4,211,693. Side-chain protecting groups, as are well known in the art, are preferably included as a part of any amino acid which has a particularly reactive side chain and optionally in the case of others, such as Trp, which amino acids are to be coupled in the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin.
Chemical intermediates made generally in accordance with the invention may be represented by the formula: X1-AA-AA2(X5)-U3-Ser(X3)-U5-U6-AA7(X2 or X7)-U8-Pro-X8 wherein: U3 is either Uxe2x80x2 or AAxe2x80x2(X2); U5 is either Uxe2x80x2 or A5(or X5); U6 is either Uxe2x80x2 or AA6(X4 or X5 or X6); U8 is either Uxe2x80x2 or AA8(X5 or X6); Uxe2x80x2 is either Lys(Xa), Aph(Xa), Orn(Xa), Dbu(Xa) or Dpr(Xa); X1 is an xcex1-amino protecting group of the type known to be useful in the art in the stepwise synthesis of polypeptides and when G in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. Among the classes of xcex1-amino protecting groups covered by X1 are (1) acyl-type protecting groups, such as formyl(For), trifluoroacetyl, phthalyl, p-toluenesulfonyl(Tos), benzoyl(Bz), benzenesulfonyl, dithiasuccinoyl(Dts) o-nitrophenylsulfenyl(Nps), tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl(Acr), chloroacetyl, acetyl(Ac) and xcex3-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl(Z), fluorenylmethyloxycarbonyl(Fmoc) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxy-carbonyl(ClZ), p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as tertbutyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as allyl(Aly), triphenylmethyl (trityl) and benzyl (Bzl); (7) trialkylsilane groups, such as trimethylsilane. The preferred xcex1-amino protecting group is Boc when X is hydrogen.
X2 is hydrogen or a protecting group for the indole nitrogen of Trp, such as Bz, Ac or For. In many syntheses there is no need to protect Trp, and such protection is not used if acylated D-Trp is present elsewhere in the peptide.
X3 is hydrogen or a protecting group for the hydroxyl side chain of Ser or Thr, e.g. Ac, Bz, trityl, DCB or benzyl ether(Bzl) and is preferably Bzl.
X4 is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, Z, 2-bromobenzyloxycarbonyl(2BrZ) and 2,6-dichlorobenzyl(DCB). 2BrZ is preferred.
X5 is a protecting group for a side chain guanidino group, such as that in Arg or Har, or for the imidazole group of His, such as nitro, Tos, trityl, adamantyloxycarbonyl, Z and 2,4-dinitrophenol(Dnp), or X5 may be hydrogen, which means there is no protection on the side chain group atoms. Tos is generally preferred.
X6 is a protecting group for an amino side chain group, primary or secondary amino, such as Z or 2ClZ; Xa is a subclass of X6 comprising such protecting groups that can be removed without removing other side chain protecting groups so as to allow the omega-amino group to thereafter take part in the reactions to build the unnatural amino-acid residue. Preferably a base-labile group, such as Fmoc, methylsulfonylethyloxycarbonyl(Msc) or trifluoroacetyl(Tfa), is used; however, it may also be possible to use a hydrazine-labile group such as phthaloyl, 
or a thiolabile group such as Nps or Dts.
X7 is hydrogen or a protecting group for Met, such as oxygen; Met is generally left unprotected.
X8 may be Gly-NH-[resin support], D-Ala-NH-[resin support] or N(A)-[resin support]; X8 may also be an amide either of Gly or of D-Ala or a substituted amide attached directly to Pro or NHNHCONH2.
The criterion for selecting side chain protecting groups for X2-X7 is that the protecting group should be stable to the reagent under the reaction conditions selected for removing the xcex1-amino protecting group (preferably Boc) at each step of the synthesis. Protecting groups generally should not be split off under coupling conditions but should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.
When the X8 group is Gly-NH-[resin support] or D-Ala-NH-[resin support], an amide bond connects Gly or D-Ala to a BHA resin or to a MBHA resin. When the X8 group is N(A)-[resin support], a substituted amide bond connects Pro to an N-alkylaminomethyl (NAAM) resin. When X8 is AzaGly-NH2, the peptide is preferably made by classical solution synthesis, as disclosed in U.S. Pat. No. 4,234,571.
When G is acetyl, for example, in the final formula, a reaction is preferably carried out with the peptide on the resin (after deblocking the xcex1-amino group while the side-chain groups remain protected), e.g. by reacting with acetic acid in the presence of diisopropyl or dicyclohexyl carbodiimide (DIC or DCC) or preferably with acetic anhydride or by another suitable reaction as known in the art.
In one aspect, the invention particularly provides a method for making a GnRH antagonist peptide having the formula:
G-AA-AA2-AA3-Ser-AA5-AA6AA7-AA8-Pro-AA10, wherein at least one of AA5 and AA6 is U* and the symbols are as set forth hereinbefore, which method comprises (a) forming an intermediate peptide having the formula:
X1-AA-AA2(X5)-AA3(X2)-Ser(X3)-Aph(Xa)-D-Aph(Xa)-AA7(X2 or X7)-AA8(X5 or X6)-Pro-X8 wherein: X1 is hydrogen or an xcex1-amino protecting group; X2 is hydrogen or a protecting group for an indole nitrogen; X3 is a protecting group for a hydroxyl group of Ser or Thr; X4 is hydrogen or a protecting group for a phenolic hydroxyl group of Tyr; X5 is either hydrogen or a protecting group for a guanidino or imidazole side chain; X6 is a protecting group for a primary amino side chain of which Xa is a subgroup that is removable without removing other protecting groups; X7 is hydrogen or a protecting group for Met; X8 is Gly-NH-[resin support] or D-Ala-NH-[resin support]; (b) removing Xa to deprotect side chain primary amino groups of the Aph residues of said intermediate peptide; (c) reacting said deprotected side chain primary amino group to build said residue into one having a triazole moiety; and (d) splitting off any remaining groups X1 to X7 and/or cleaving from resin support included in X8.
Purification of the peptide is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution system: n-butanol; 0.1N acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25, or by using HPLC, as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography, 288 (1984) 303-328.
The antagonists of the invention are effective at levels of less than 100 micrograms per kilogram of body weight, when administered subcutaneously at about noon on the day of proestrus, to prevent ovulation in female rats. For prolonged suppression of ovulation, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are particularly soluble at physiological pHs and thus can be prepared as relatively concentrated solutions for administration. The antagonists are also effective to arrest spermatogenesis when administered to male mammals on a regular basis and can thus be used as contraceptives. Since these compounds will reduce testosterone levels (an undesired consequence in the normal, sexually active male), it may be reasonable to administer replacement dosages of testosterone along with the GnRH antagonist. These antagonists can also be used to regulate the production of gonadotropins and sex steroids for other purposes as indicated hereinbefore.
In the following formulas, the U* residues are defined in terms of the original amino acid residue having a side chain amino group plus the modification in question which is set forth in the accompanying parentheses. Preferably, the original residue is incorporated in the main peptide chain, for example Lys or D-Lys or Orn, Dbu, Dpr or a D-isomer thereof, and is modified while a part of the peptide chain that is still attached to the resin to form the desired residue of the amino acid U*. However, as indicated hereinbefore, the suitably protected unnatural amino acid U* can be added as a part of the usual chain elongation process.
With respect to modified side chain amino groups of the amino acids Lys, Orn, Dbu and Dpr, the following abbreviations are used:
act=acetyl aminotriazole
bcg=aminobutyl cyanoguanidino
bzcg=aminobenzyl cyanoguanidino
bur=N-g-amido, N-gxe2x80x2-butylguanidino
chcg=aminocyclohexyl cyanoguanidino
ecg=aminoethyl cyanoguanidino
icg=aminoisopropyl cyanoguanidino
hcg=aminohexyl cyanoguanidino
hicg=histaminyl cyanoguanidino (ethylimidazole)
mcg=aminomethyl cyanoguanidino
ncg=aminoethyl(1 or 2)naphthyl cyanoguanidino
mncg=aminomethyl(1 or 2)naphthyl cyanoguanidino
Ocg=O-phenyl cyanoguanidino
pcg=aminopropyl cyanoguanidino
Sbcg=thiobutyl cyanoguanidino
tcg=3-amino 1,2,4 triazole
trcg=indole ethylamino cyanoguanidino(tryptamino cyanoguanidino)
mpcg=aminomethyl pyridyl cyanoguanidino (number indicates position of aminomethyl group on pyridyl ring)